The role of p53 family members in epithelial lineage establishment and maintenance Project summary p53 is a transcription factor that plays a fundamental role in tumor suppression and is often mutated in cancer. p63, on the other hand, is exclusively expressed in epithelial cells and is rarely mutated in cancer. While deletion of Trp53 (encoding the p53 protein) shows no phenotype at birth, deletion of Trp63 (encoding the p63 protein) in mice leads to clear developmental and morphological defects in the squamous epithelia and epidermis. In humans, germline mutations in p63 cause ectodermal dysplastic syndromes, leading to cleft palate and limb malformations. In order to improve and develop novel treatments for epithelial cancers and p63 related genetic disorders, our understanding of their role in epithelial lineage establishment and maintenance needs to be largely expanded. In particular, the role of p53 family members in de novo epithelial lineage commitment and the molecular and epigenetic mechanisms by which they maintain epithelial cell identity though enhancer regulation and pioneer factor activity remain to be elucidated. Our lab previously showed that 40% of p53 binding sites in the fibroblast lineage are located at sites with a closed chromatin conformation and devoid of promoter or enhancer specific histone modifications. Further analysis strongly suggested that these nearly 2000 locations adopt an open chromatin conformation, decorated by enhancer specific marks (H3K4me and H3K27ac) specifically in epithelial cells. Furthermore, both p53 and p63 bind directly to these sites in epithelial lineages. Based on our lab's previous discoveries and expertise I propose to: (1) Dissect the role of p53 family members in epithelial enhancer establishment and epithelial lineage commitment. To this end, I will utilize a novel temporally-controlled fibroblast to keratinocyte reprogramming system. This versatile system coupled with genomic and biochemical tools will allow me to study the role p63 isoforms and mutants, as well as p53's role in de novo establishment of epithelial identity. (2) Determine how p63 maintains epithelial identity. Here, I will utilize CRISPR Cas9 on normal human epidermal keratinocytes to introduce mutations in p63 and in p63 response elements at specific genomic loci. This will allow me to directly observe how mutations in p63 and loss of p63 binding contribute to loss of epithelial identity. Together these aims will provide novel insight into the roles of p53 family members in epithelial lineage establishment and maintenance, as well as molecular mechanisms related to disease. 1